Stereoselective antifibrillogenic peptides and peptidomimetics thereof

ABSTRACT

The present invention relates to antifibrillogenic agents for inhibiting amyloidosis and/or for cytoprotection for the treatment of amyloidosis disorders. These agents include peptides, isomers thereof and peptidomimetic compounds thereof. These agents comprise a peptide having a sequence identified from the glycosaminoglycan (GAG) binding region and the prot-prot interaction region of the amyloid protein. The peptide has at least one [D] amino acid isomer substitution. The invention also relates to the peptide bound to a label for in vivo imaging of amyloid deposits.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/009,122, filedJun. 18, 2002 (pending), which claims priority under 35 U.S.C. § 371from PCT/CA00/00515, filed May 4, 2000, which claims priority from U.S.Ser. No. 60/132,592, filed May 5, 1999, the contents of each of whichare incorporated herein by reference.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The invention relates to agents having potent antifibrillogenic activityfor the treatment of amyloidosis disorders and for imaging of amyloiddeposits. These agents include peptides and peptidomimetic compoundsthereof.

(b) Description of Prior Art

Amyloidosis refers to a pathological condition characterized by thepresence of amyloid fibers. Amyloid is a generic term referring to agroup of diverse but specific extracellular protein deposits that areseen in a number of different diseases. Though diverse in theiroccurrence, all amyloid deposits share common morphologic properties,stain with specific dyes (e.g. Congo red), and have a characteristicred-green birefringent appearance in polarized light after staining.They also share common ultrastructural, x-ray diffraction and infraredspectra features.

Some amyloidotic diseases can be idiopathic but most of these diseasesappear as a complication of a previously existing disorder. For example,primary amyloidosis can appear without any other pathology or can followplasma cell dyscrasia or multiple myeloma. Secondary amyloidosis isusually seen associated with chronic infection (such as tuberculosis) orchronic inflammation (such as rheumatoid arthritis). A familial form ofsecondary amyloidosis is also seen in Familial Mediterranean Fever(FMF). This familial type of amyloidosis, as one of the other types offamilial amyloidosis, is genetically inherited and is found in specificpopulation groups. Isolated forms of amyloidosis are those that tend toinvolve a single organ system. Different amyloids are also characterizedby the type of protein present in the deposit. For example,neurodegenerative diseases such as scrapie, bovine spongiformencephalitis, Creutzfeldt-Jakob disease and the like are characterizedby the appearance and accumulation of a protease-resistant form of aprion protein (referred to as AScr or PrP-27) in the central nervoussystem. Similarly, Alzheimer's disease, another neurodegenerativedisorder, is characterized by congophilic cerebral angiopathy, neuriticplaques and neurofibrillary tangles. In this case, the plaque and bloodvessel amyloid is formed by the deposition of fibrillar Aβ amyloidprotein. In adult-onset diabetes, amyloids containing the IAPP amyloidprotein accumulate in the pancreas. Other systemic diseases,complications of long-term hemodialysis and sequelae of long-standinginflammation or plasma cell dyscrasias are characterized by theaccumulation of amyloids systemically. In each of these cases, adifferent amyloidogenic protein is involved in amyloid deposition.

Once these amyloids have formed, there is no known, widely acceptedtherapy or treatment that significantly dissolves the deposits in situ.

Each amyloidogenic protein has the ability to organize into β-sheet andto form insoluble fibrils that get deposited extracellularly. Eachamyloidogenic protein, although different in amino acid sequence has thesame property of forming fibrils and binding to other elements such asproteoglycan (glycosaminoglycan), amyloid P and complement component.Moreover, each amyloidogenic protein has amino acid sequences, which,although different, will show similarities such as regions with theability to bind to GAG's (referred to as the GAG binding site) as wellas other regions that will promote β-sheet formation referred to asβ-sheet region.

In specific cases, amyloidotic fibrils once deposited can become toxicto the surrounding cells. As per example, the Aβ fibrils organized assenile plaques have been shown to be associated with dead neuronal cellsand microgliosis in patients with Alzheimer's disease. When tested invitro, Aβ peptide was shown to be capable of triggering an activationprocess of the microglia (brain macrophages), which would explain thepresence of microgliosis and brain inflammation found in the brain ofpatients with Alzheimer's disease.

In another type of amyloidosis seen in patients with Type II diabetes,the amyloidogenic protein IAPP, has been shown to induce β-islet celltoxicity in vitro. Hence, appearance of IAPP fibrils in the pancreas ofType II diabetic patients could contribute to the loss of the β isletcells (Langerhans) and organ dysfunction.

Particularly, in patients with Alzheimer's Disease, an agent capable 1)of preventing amyloid fibril formation and deposition and 2) of directlyor indirectly inhibiting Aβ-induced neurotoxicity and inflammation(microgliosis), could be a treatment of choice to prevent and arrest thedevelopment of Alzheimer's disease.

It would be highly desirable to be provided with agents having potentantifibrillogenic activity for the treatment of amyloidosis disorders.

SUMMARY OF THE INVENTION

One aim of the present invention is to provide agents having potentantifibrillogenic activity for the treatment of amyloidosis disorders.

Another aim of the present invention is to provide a method for thetreatment of amyloidosis disorders, such as Alzheimer's' disease.

A number of strategies for possible therapeutic intervention in amyloiddevelopment have been proposed. These strategies include reduction ofthe pool of precursor proteins, prevention of the interaction ofprecursor proteins and disruption of preformed amyloid. The presentinvention deals mainly with the second approach, prevention of precursorprotein interactions. The ideal molecule to fulfill this function, wouldinteract specifically with the amyloid protein and would in so doingprevent the protein from interacting with itself. When dealing withmolecules that are chiral, it is standard practice to identify which ofthe stereoisomers possesses the activity, since in general, activity canbe attributed to one or the other of the isomers. By using astereochemically pure isomer, side reactions can be avoided or reduced.

In accordance with one embodiment of the present invention there isprovided an antifibrillogenic agent for inhibiting amyloidosis and/orfor cytoprotection, which comprises a peptide of Formula I, an isomerthereof, a retro or a retro-inverso isomer thereof or a peptidomimeticthereof:Xaa₁-Xaa₂-Xaa₃-Xaa₄  Iwherein,Xaa₁ is absent or selected from the group consisting of Lys, Lys-Lys,Xaa₅-Lys-, and Ala;Xaa₅ is absent or selected from the group consisting of His-Gln-,His-His-Gln-, Val-His-His-Gln-, Glu-Val-His-His-Gln-, Asp-Asp-Asp-,Lys-Val-Asp-Asp-Gln-Asp-, Gln-;Xaa₂ is absent or any amino acid;Xaa₃ is absent, Val or Phe;Xaa₄ is absent or selected from the group consisting of Phe, Phe-NH₂,Phe-Phe, Phe-Phe-Ala, Phe-Phe-Ala-NH₂, Phe-Phe-Ala-Gln,Phe-Phe-Ala-Gln-NH₂, Val-Leu-Lys, Val-Leu-Lys-NH_(2;)wherein the peptide of formula I contains at least one Lys or Asp;and wherein the peptide has at least one [D] amino acid residue,with the proviso that Lys-Lys-Leu-Val-Phe-Phe-Ala is an all-[D] peptide;and with the proviso that when Xaa₅ is Lys-Val-Asp-Asp-Gln-Asp- all ofXaa₂, Xaa₃, and Xaa₄ are absent.

Preferably, Xaa₂ is a hydrophobic amino acid residue such as a leucineresidue.

In one embodiment of the invention, the peptide of formula I has atleast two [D] amino acid residues, and more preferably at least three[D] amino acid residues. Optionally, the peptide of formula I has one[L] amino acid residue, or more preferably the peptide is an all-[D]isomer peptide.

In another embodiment of the invention, the peptide of Formula I isselected from the group consisting of: Lys-Ile-Val-Phe-Phe-Ala; (SEQ IDNO:1) Lys-Lys-Leu-Val-Phe-Phe-Ala; (SEQ ID NO:2)Lys-Leu-Val-Phe-Phe-Ala; (SEQ ID NO:3) Lys-Phe-Val-Phe-Phe-Ala; (SEQ IDNO:4) Ala-Phe-Phe-Val-Leu-Lys; (SEQ ID NO:5) Lys-Leu-Val-Phe; (SEQ IDNO:6) Lys-Ala-Val-Phe-Phe-Ala; (SEQ ID NO:7) Lys-Leu-Val-Phe-Phe; (SEQID NO:8) Lys-Val-Val-Phe-Phe-Ala; (SEQ ID NO:9)Lys-Ile-Val-Phe-Phe-Ala-NH₂; (SEQ ID NO:10) Lys-Leu-Val-Phe-Phe-Ala-NH₂;(SEQ ID NO:11) Lys-Phe-Val-Phe-Phe-Ala-NH₂; (SEQ ID NO:12)Ala-Phe-Phe-Val-Leu-Lys-NH₂; (SEQ ID NO:13) Lys-Leu-Val-Phe-NH₂; (SEQ IDNO:14) Lys-Ala-Val-Phe-Phe-Ala-NH₂; (SEQ ID NO:15)Lys-Leu-Val-Phe-Phe-NH₂; (SEQ ID NO:16) Lys-Val-Val-Phe-Phe-Ala-NH₂;(SEQ ID NO:17) Lys-Leu-Val-Phe-Phe-Ala-Gln; (SEQ ID NO:18)Lys-Leu-Val-Phe-Phe-Ala-Gln-NH₂; (SEQ ID NO:19)His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala- (SEQ ID NO:20) NH₂; Asp-Asp-Asp;(SEQ ID NO:21) Lys-Val-Asp-Asp-Gln-Asp; (SEQ ID NO:22) His-His-Gln-Lys;(SEQ ID NO:23) and Gln-Lys-Leu-Val-Phe-Phe-NH₂. (SEQ ID NO:24)

More preferably, the peptide of formula I is a peptide as set forth inSEQ ID NO:2 or SEQ ID NO:3.

In accordance with one embodiment of the present invention there isprovided a labeled conjugate for in vivo imaging of amyloid plaque,which comprises a conjugate of formula II:A-B-C  IIwherein A is an amyloid plaque-targeting moiety selected from the groupconsisting of a peptide of Formula I as defined above, an isomerthereof, a retro or a retro-inverso isomer thereof and a peptidomimeticthereof,wherein B is a linker portion allowing attachment of the amyloidplaque-targeting moiety to C; andwherein C is a label that allows for in vivo imaging.Preferably, the linker portion B is selected from the group consistingof Glucose and Phe. Preferably, the label C is ^(99m)Tc.

Still in accordance with the present invention, there is provided amethod for the treatment of amyloidosis disorders in a patient, whichcomprises administering to the patient a therapeutically effectiveamount of a peptide of Formula I, or the antifibrillogenic agent, asdefined above.

Further in accordance with the present invention, there is provided acomposition for the treatment of amyloidosis disorders in a patient,which comprises a therapeutically effective amount of a peptide ofFormula I, or of an antifibrillogenic agent, as defined above inassociation with a pharmaceutically acceptable carrier.

In accordance with the present invention, there is also provided acomposition for in vivo imaging of amyloid plaques, which comprises atherapeutically effective amount of a labeled conjugate as defined abovein association with a pharmaceutically acceptable carrier.

The peptide of Formula I or the antifibrillogenic agent may be used forinhibiting amyloidosis and/or for cytoprotection.

The labeled conjugate may be used for in vivo imaging of amyloidplaques.

The peptide of Formula I or the antifibrillogenic agent mayalternatively be used for the manufacture of a medicament for inhibitingamyloidosis and/or for cytoprotection.

Similarly, the labeled conjugate may also be used for the manufacture ofa medicament for in vivo imaging of amyloid plaques.

Other embodiments of these peptides include racemic mixtures andpeptides having mixed chirality, i.e., different chirality at differentchiral centers.

In accordance with the peptides Lys-Lys-Leu-Val-Phe-Phe-Ala (SEQ IDNO:2) and Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:3), one stereoisomer, the Dform, is found to be more active than the L form, and the D isomer isthe preferred form for use of this peptide as a drug.

The present invention further provides similar peptides designed for theother amyloidogenic peptides such as AA, AL, and IAPP. In fact, thepresent invention also provides a peptide for inhibiting amyloidosisand/or for cytoprotection. The peptide has a sequence taken from theβ-sheet region of an amyloid protein. Such peptide or a compositioncontaining such peptide can be used for inhibiting amyloidosis and/orfor cytoprotection. Alternatively, such peptide or a compositioncontaining such peptide can be used for the manufacture of a medicamentfor inhibiting amyloidosis and/or for cytoprotection.

Accordingly, the present invention also provides a composition forinhibiting amyloidosis and/or for cytoprotection, which comprises atherapeutically effective amount of a peptide as defined previously inassociation with a pharmaceutically acceptable carrier.

In accordance with the present invention, the amyloidosis disorderincludes, without limitation, prion protein related disorders, type IIdiabetes and Alzheimer's disease.

With regard to another aspect of the invention, diseases caused by thedeath or malfunctioning of a particular type or types of cells can betreated by transplanting into the patient healthy cells of the relevanttype of cell. Often these cells are cultured in vitro prior totransplantation to increase their numbers, to allow them to recoverafter the isolation procedure or to reduce their immunogenicity.However, in many instances the transplants are unsuccessful, due to thedeath of the transplanted cells. The inventors have now also found thatculturing of cells can lead to the formation of fibrils from endogenousproteins. Such fibrils are likely to continue to grow after the cellsare transplanted and cause death or dysfunction of the cells. Theinventors have also found that the peptide of the present invention orthe antifibrillogenic compound of the present invention can be used toreduce the formation of fibrils.

Thus the invention also provides a process for the preparation of cellssuitable for transplantation into a mammal, which cells are capable offorming fibrils. The process comprises contacting the cells with thepeptide of the present invention or the antifibrillogenic compound ofthe present invention.

The peptide of Formula I or the antifibrillogenic compound causesbreakdown of amyloid deposits which have been formed by the cells priorto the contact. Preferably, the cells are cultured in the presence ofthe peptide of Formula I or the antifibrillogenic compound.

For the purpose of the present invention the following expressions andterms are defined below.

The term “agents having stereoselective antifibrillogenic activity” isintended to mean any peptides, peptide analogues, peptide derivatives,or peptidomimetics that retain the stereoselective antifibrillogenicactivity, the cytoprotective and anti-inflammatory activity and/or theability to alter a natural amyloidotic protein aggregation as describedherein. Peptide analogues, peptide derivatives, or peptidomimeticsinclude any molecules that mimic the chemical structure of a peptide andretain the functional properties of the peptide (Williams, W. V. andWeiner, D. B., eds., Biologically Active Peptides: Design, Synthesis,and Utilization, vol. 1, Technomic Publishing Company Inc., Lancaster,Pa., 1993, pages 35-3 . . . ). Examples of peptide analogues, peptidederivatives, or peptidomimetics include compounds with sulfonamide,phosphoramide or non-amide linkages.

The expression “antifibrillogenic activity” is intended to mean theability to block or prevent an amyloidogenic protein from formingfibrils, preferably by preventing it from adopting its β-pleatedconformation.

The term “cytoprotection” or “cytoprotective activity” is intended tomean the ability to protect cells from amyloid-induced toxicity.

The expression “anti-inflammatory” is intended to mean the ability toblock or reduce the Aβ-induced microglial activation process or to blockthe chemokine-induced inflammatory reaction.

The expression “retro isomer” is intended to mean a reversal of thedirection of the peptide backbone.

The expression “inverso isomer” is intended to mean an inversion of theamino acid chirality used to make the peptide.

The expression “retro-inverso isomer” is intended to mean a reversal ofboth the peptide backbone direction and the amino acid chirality.

Except as otherwise expressly defined herein, the abbreviations usedherein for designating the amino acids and the protective groups arebased on recommendations of the IUPAC-IUB Commission on BiochemicalNomenclature (Biochemistry, 1972, 11:1726-1732).

Also, unless specified otherwise, the Aβ(1-40) is the naturallyoccurring Aβ(1-40), that is the all [L]-isomer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the targeted sites of the protein-proteininteractions required for self-assembly into β-sheet fibrils;

FIG. 2 illustrates a thioflavin T fluorescence assay for fibrilformation by [L]-Aβ (1-40) in the absence and presence of a peptide inaccordance with one embodiment of the invention;

FIG. 3 shows the same assay as in FIG. 2 for fibril formation by [D]-Aβ(1-40);

FIG. 4 is a bar graph illustrating the percentage of thioflavin Tfluorescence in the presence of the [D]-peptide used in FIG. 2, with orwithout single substitutions of corresponding [L]-amino acids;

FIG. 5 is a bar graph illustrating a thioflavin T fluorescence assay forfibril formation by [L]-Aβ (1-40) in the presence of the [D]-peptideused in FIG. 2, with or without substitution of the Leu residue by otherhydrophobic amino acids;

FIG. 6 illustrates the toxicity of [L]-Aβ (1-40) in the absence andpresence of peptides in accordance with one embodiment of the invention;and

FIG. 7 is a bar graph illustrating the toxicity of [L]-Aβ (1-40) in thepresence of another peptide of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

As illustrated in FIG. 1, internal regions of the Aβ sequence have beenshown to confer characteristics of the amyloid protein. Indeed, theregion between amino acid 13-16 (His-His-Gln-Lys, SEQ ID NO:23) of theamyloid protein is responsible for the interaction between the Aβprotein and the glycosaminoglycan moiety of the proteoglycans(Kisilevsky, R., et al., Proteoglycans and amyloid fibrillogenesis: Thenature and origin of amyloid fibrils, Wiley, Chichester (CIBA FoundationSymposium 1997), pp. 58-72). Proteoglycans are known to promote amyloidfibril formation as well as protect these fibrils from proteolysis(Gupta-Bansal, R., et al., 1995, The Journal of Biological Chemistry,270:18666-18671). More recently, the same region has been determined toplay a role in the activation process of microglial cells by Aβ(Giulian, D., et al., 1998, The Journal of Biological Chemistry,273(45):29719-29726). This 13-16 region of Aβ, often referred to as theGAG binding site, is also part of a larger domain, the 10-16 region ofthe protein which has been suggested as the region responsible for theadherence of Aβ to the cell surface (Giulian, D., et al., 1996, TheJournal of Neuroscience, 16(19):6021-6037). Such adherence of Aβ to thecell surface will allow the interaction of Aβ with the specific cellsleading to either microglia activation or toxicity of neuronal cells.

These two overlapping regions of the Aβ protein, i.e. amino acids 13-16and 10-16 are adjacent to the 16-21 region of Aβ, a short hydrophobicstretch critical for the formation of fibrillar structures (Hilbrich,C., et al., 1992, J. Mol. Biol., 228:460-473). By having peptidescapable of interacting with these overlapping regions of Aβ, one can aimat preventing both Aβ fibril formation and Aβ cellular interaction (i.e.microglia activation, neurotoxicity).

A preferred embodiment of the present invention is novel and arises fromthe unexpected finding that the all-[D] stereoisomer peptides,Lys-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:2) and Lys-Leu-Val-Phe-Phe-Ala(SEQ ID NO:3), are much more potent inhibitors of Aβ(1-40)fibrillogenesis then the corresponding all-[L] peptides. The all-[D]stereoisomer peptides, Lys-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:2) andLys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:3) are also potent cytoprotectiveagents.

This finding was unforeseen particularly because the researchers whooriginally reported peptides containing the sequenceLys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:3) as an inhibitor offibrillogenesis, state in a second article which they published: “Apeptide entirely composed of amino acids in D configuration with thesequence klvff (lowercase marks amino acids in D configuration) wassynthesized using the SPOT technique and assayed for ¹²⁵I-LBMP1620binding. This peptide failed to bind ¹²⁵I-LBMP1620 indicating thatKLVFF-KLVFF interaction is sterospecific.” Tjernberg, L. O. et al.(1997) Controlling Amyloid β-Peptide Fibril Formation withProtease-stable Ligands, J. Biol. Chem., 272:12602.

Inhibition of Amyloidosis

The experimental work performed leading to this invention includedcomparing the ability of the [D] and [L] stereoisomers of peptideLys-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:2) to inhibit the fibrillogenesisprocess observed with the amyloidogenic peptide Aβ(1-40) in a thioflavinT fluorescence assay.

The thioflavin T fluorescence assay for fibrillogenesis is based on theprinciple that the fluorescent dye, thioflavin T, binds specifically tofibrillar, but not to unaggregated Aβ peptide (LeVine III, H., 1993,Protein Science 2:404-410). Upon binding, thioflavin T develops acharacteristic fluorescence (Naiki, H., et al., 1996, Lab. Invest. 74:374-383), which can be easily detected. The dye is believed to interactwith the stacked cross-β pleated sheets, the common structural motif ofall amyloids (LeVine III, H., 1995, Amyloid: Int. J. Exp. Clin Invest.2:1.6). Thioflavin T is widely used to assay the effect of compounds onAβ peptide fibrillogenesis (Bronfman, P. C., et al., 1995, NeuroscienceLett. 218:201-203).

In this assay test compounds are incubated with a solution of Aβ(1-40)(20 μM) containing 10 μM thioflavin T, in 0.02M Tris/0.02M acetate/0.15MNaCl/0.005% azide/pH 7.40 at 37° C. in sealed 384 well microplates.Readings (ex 430 nm/em 485 nm) are taken at various time intervals witha microplate fluorescence reader. An increase in fluorescence signifiesthe appearance of amyloid or intermediates in the production of amyloid.Inhibitors of fibrillogenesis will lead to less fluorescence production.

The results illustrated in Table 1 below, are based on the fluorescenceproduction in the presence of test peptides at either 20 μM or 80 μMconcentration, at the time intervals of 5, 19, 45, 67, 77 and 90 hours,compared to a control, buffer alone, without added inhibitory peptide.TABLE 1 Order Of Potency of Peptide Inhibitors Tested at Tested at 20 μM80 μM (strongest activity) 1 (D) KIVFFA 1 (D) AFFVLK 2 (D) KKLVFFA 1 (D)KKLVFFA 3 (D) KLVFFA 1 (D) KLVFFA 4 (D) KFVFFA 1 (D) KFVFFA 5 (D) AFFVLK5 (D) KIVFFA 6 (D) KLVF 6 (D) KAVFFA 7 (D) KAVFFA 7 (L) KKLVFFA 8 (L)KLVFFA 8 (L) KLVFFA 9 (D) KLVFF 9 (D) KLVF 10 (L) KKLVFFA 10 (D) KLVFF(weakest activity) 11 (L) AFFVLK 11 (L) AFFVLKProtocolAβ peptide: Aβ(1-40) 95% purity (American Peptide Company, Inc,Sunnyvale, Cal. USA, cat. 62-0-78) is disaggregated in trifluoroaceticacid and filtered through a 0.02 μM filter, (Whatman Anotop 25 plus,0.02 μm, Catalogue no. 6809 4102) in hexafluoroisopropanol (HFIP).Solutions of Aβ(1-40) at 600 μM in HFIP are stored at −80° C.Assay mixture: The mixture is prepared as two solutions that arecombined upon addition to the 384 well microplate (Corning Costar cat.3705).

-   i) Solution A consists of test peptides in 0.02M Tris/0.02M    acetate/0.15M NaCl/0.01% azide at pH 7.40 or buffer alone (control),-   ii) Solution B consists of Aβ(1-40) 40 μM, thioflavin T 20 μM in    0.02M Tris/0.02M acetate/0.15M NaCl at pH 7.40. This solution is    prepared by drying the Aβ peptide under nitrogen and then    resuspending this in 0.04M Tris base with 15 minutes sonication. An    equal volume of 0.04M acetic acid containing 0.3 M NaCl is added and    the solution is adjusted to pH 7.40±0.02. A small volume of 5 mM    thioflavin T is added to the solution to give a final 20 μM    concentration of thioflavin T.-   iii) The microplate is loaded with 40 μL of solution A followed by    40 μL of solution B which gives a final 20 μM Aβ(1-40), 10 μM    thioflavin T, and either 20 μM, 80 μM or 100 μM test compound in    0.02M Tris/0.02M acetate/0.15M NaCl/0.005% azide, pH 7.40. The plate    is sealed and loaded into the microplate fluorescence reader.    Fluorescence measurement data analysis: The HTS-7000 Bio Assay    Reader, Perkin Elmer, is used to perform kinetic runs of about 5    days. Readings were taken at various time intervals, 5, 19, 45, 67,    77 and 90 hours, with one minute shaking before each reading.    Bandpass filters used were: excitation 430 nm, emission 485 mm.    Calculations

The rank order of efficacy of the peptides is determined by observingwhich peptides allow the appearance of fluorescence, above thebackground level, first. For example in the presence of buffer controlalone, fluorescence appears earlier than when any of the peptides ispresent. The most active peptides prevent the appearance of fluorescenceeven after 90 hours of incubation.

The results achieved in the thioflavin T fibrillogenesis assays showthat all-[D] stereoisomer peptide was about 60 times more potent thenthe all-[L] stereoisomer peptide. This is based on the observation that400 μM all-[L] stereoisomer was required to give an equivalentinhibition to that produced with 6.1 μM all-[D] stereoisomer peptide.

The results achieved in the Aβ-NBD environmental probe fibrillogenesisassay showed that the all-[D] stereoisomer peptide was at least 30 timesmore potent than the all-[L] stereoisomer peptide. This estimate isbased on the observation that the lowest concentration of all-[D]peptide tested (25 μM) was more potent than the highest concentration ofthe all-[L] peptide (800 μM).

β-Sheet and GAG Binding Domains Peptides

Novel peptides and peptidomimetics that include complementary sequencesto certain portions of amyloidogenic peptides such as Aβ, AA, AL, IAPP,and prion proteins are designed to be capable of inhibition ofProtein-Protein interactions or self assembly. The targeted portions inthe various disease-causing proteins aforementioned, preferably containone or more charged residues such as aspartate, glutamate, lysine,histidine and arginine. Such peptides and their peptidomimetics willinhibit fibrillogenesis of the amyloidogenic peptides and prion proteinsand interfere with chemokines binding to the cell surface proteoglycansleading to dimerization or tetramerization by interacting with their GAGbinding domains. In the case of Aβ, these interactions lead tocytoprotection as well as inhibition of inflammatory response and serveas potent therapeutics for the treatment of Alzheimer's disease. In thecase of chemokine-related disorders these interactions may lead to adecrease in the uncontrolled inflammatory response associated with somediseases.

Other amyloidogenic peptides such as IAPP, have also been tested. Forexample, 2 peptides from the β-sheet region of IAPP have been shown toinhibit IAPP fibril formation using the thioflavin T fluorescence assay,circular dichroism (measures secondary structure) and the electronmicroscope (to look at fibrils directly).

The full-length IAPP is 37 amino acids and the β-sheet region is the20-29 sequence. The 20-29 sequence is critical for forming β-sheet andhas been previously shown to be a key region in modulating IAPPaggregation and folding. Hexapeptides from this β-sheet region wereexamined and 2 were found to be active.

Hexapeptides spanning the 20-29 region(Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser) of the IAPP protein weresynthesized and tested for their ability to prevent fibril formation asdetermined by circular dichroism and the thioflavin T assay.Hexapeptides were designed and were found to be capable of blocking theformation of IAPP fibrils. These peptides (Ser-Asn-Asn-Phe-Gly-Ala- andAsn-Asn-Phe-Gly-Ala-Ile) were directed towards the central core of the20-29 region.

Novel peptides containing 3-6 residues that are complementary (in termsof their charges) to the 10-16 segment of Aβ peptide have been shown forthe first time to strongly interact with Aβ peptide. They provide astarting point for the design of BBB (blood brain barrier) permeablepeptidomimetics. In principle, the present invention provides similarpeptides can be designed for the other amyloidogenic peptides such asAA, AL, and IAPP.

Asp-Asp-Asp (SEQ ID NO:21), a tripeptide, when incubated with Aβ40 underphysiological conditions shows a slight decrease at time t=0 in theamount of β-sheet content as is evident by the CD spectrum. Incubationof this tripeptide with Aβ40 for 24 hours shows no trace of β-sheetconformation of the Aβ40 and clearly indicates the ability of thistripeptide to strongly interact with Aβ40 peptide and keep Aβ40 in arandomized and non-fibrillary conformation. The anti-fibrillogenicproperty of this tripeptide is also supported by the Aβ42 solubilizationassay.

Lys-Val-Asp-Asp-Gln-Asp (SEQ ID NO:22), a hexapeptide, when incubatedwith Aβ40 under physiological conditions shows an increase at time t=0in the amount of β-sheet content as is evident by the CD spectrum.Incubation of this hexapeptide with Aβ40 for 24 hours shows a dramaticincrease in β-sheet content of the Aβ40 and clearly indicates theability of this hexapeptide to strongly interact with Aβ40 peptide andorganize it into a β-sheet conformation. Electron microscopy of themixture failed to show any fibrils indicating that this particularcompound is in fact an anti-fibrillogenic compound with regard to Aβ. Invitro results with NBD and thioflavin-T based fluorescence assaysconfirm this finding. It is the understanding of the inventors that thisinteresting observation will lead to a greater understanding offibrillogenesis of Aβ40 and Aβ42 peptides and as a result, will provideimportant information for the design of potent anti-fibrillogeniccompounds for Aβ, other amyloidotic peptides such as AA, AL and IAPP forthe treatment of diseases such as Alzheimer's, Type II Diabetes andamyloidosis related disorders. The same principle can also be applied tothe design of peptide type compounds for the inhibition of binding ofvarious chemokines to the cell surface as well as inhibition ofself-assembly and cellular adherence of prion proteins.

The results illustrated in FIG. 2 show that all[D]-Lys-Leu-Val-Phe-Phe-Ala (SEQ. ID NO: 3) is a more potent inhibitorof Aβ (1-40) assembly in the thioflavin T fluorescence assay than is all[L]-Lys-Leu-Val-Phe-Phe-Ala. Since the naturally occurring Aβ (1-40)used in these experiments was the all-[L] amino acid version, theseresults indicate that an inhibitor peptide works best when containingamino acids of the opposite chirality.

FIG. 3 demonstrates that the same rule of opposite chirality illustratedin FIG. 2 applies for the assembly of Aβ (1-40) synthesized using aminoacids of the [D] type. In this experimentall-[L]-Lys-Leu-Val-Phe-Phe-Ala (SEQ. ID NO:3) is a more potentinhibitor in the all-[D]-Aβ (1-40) assembly reaction thanall-[D]-Lys-Leu-Val-Phe-Phe-Ala. This result confirms that peptides ofopposite chirality are better inhibitors.

FIG. 4 illustrates the inhibition of Aβ (1-40) fibril formation byall-[D]-Lys-Leu-Val-Phe-Phe-Ala (20 μM) with or without singlesubstitutions of [L]-amino acids in the thioflavin T fluorescence assay.In this experiment the ability of the all-[D]-Lys-Leu-Val-Phe-Phe-Alapeptide to inhibit Aβ (1-40) fibril formation, measured as percentage ofthioflavin T fluorescence in the absence of peptide (control), wascompared to [D]-Lys-Leu-Val-Phe-Phe-Ala peptides with single [L]-aminoacid replacements. None of the mixed chirality Lys-Leu-Val-Phe-Phe-Alapeptides were more potent than the original all-[D] peptide. This resultdemonstrates that [D]-amino acids are more potent inhibitors of Aβ(1-40) fibrillogenesis than [L]-amino acids.

However as seen in FIG. 4 some peptides with single [L] substitutions doretain inhibitory activity. In particular peptides in which the [D]isomer of the Lys, the second Phe and the Ala are substituted with the[L]-isomers retain inhibitory activity. The substitutions, which reduceinhibitory activity the most, are the Leu, the Val and the first Phe,indicating that these residues contribute the most to the potency of the[D]-peptide. From FIG. 4, it is apparent that peptides with mixedchirality or with at least one [D]-substituted amino acid are alsoinhibitors, although not as potent as the all-[D] peptide. Thesemixed-chirality peptides are thus meant to be included in the presentinvention.

FIG. 5 illustrates the inhibition of Aβ (1-40) fibril formation in thethioflavin T fluorescence assay by all-[D]-Lys-Leu-Val-Phe-Phe-Ala (20μM), with or without replacement of the leucine by other hydrophobicamino acids. In this experiment all the peptides tested retained someinhibitory activity. This result demonstrates that the leucine residueis not critical for inhibition of Aβ fibril formation in the all-[D]peptide. These results illustrated in FIG. 5 were non-obvious andunexpected in light of a prior publication which identified the Leucineresidue as critical in an all-[L] version of the peptide (Tjernberg L Oet al., J. Biol. Chem. 271:8545, 1996).

Cytoprotection

The experimental work performed leading to this invention also includedcomparing the ability of [D] and [L] stereoisomers of the peptides ofthe present invention to show cytoprotective activity, i.e. to protectcells from Aβ toxicity.

FIG. 6 uses the MTT assay on SH-SY5Y cells.

Protocol

A SH-SY5Y human neuroblast cell line (American Type Culture Collection,cat. CRL-2266) is cultured according to technical specifications.Monomerized Aβ (1-40) is prepared using trifluoroacetic acid andhexafluoroisopropanol, in the same way already described for thethioflavin T fluorescence assay. Monomerized Aβ at variousconcentrations in TANA buffer (0.02 M TRIS base pH 7.4, 0.02M acetate,0.15 M NaCl) is added to 100 μM test peptide and the mixture isincubated for 24 hours at 37° C. with agitation, in order to allowpolymerization to occur. Cells are adhered to a 96-well microplate for 2hours at 37° C. and 5% CO₂ before the Aβ-peptide mixture, or bufferalone (control), is added. The microplate is gently agitated andincubated for 20-24 hours at 37° C. and 5% CO₂. Cell viability isdetermined by a MTT-based colorimetric assay. The MTT assay (BoehringerMannheim, Cell Proliferation Kit 1) is based on the principle that theyellow tetrazolium salt MTT is cleaved in metabolically active cells toproduce purple formazan crystals. The formazan crystals are solubilizedand the resulting colored solution is quantified using a scanningmultiwell spectrophotometer (ELISA reader, Absorbance A₅₆₀ nm). Cellulartoxicity was calculated as follows:${{Toxicity}\quad(\%)} = {100 - {\frac{\left( {{O.D\quad.\quad{sample}} - {O.D.\quad{Blank}}} \right)}{\left( {{O.D.\quad{Control}} - {O.D.\quad{Blank}}} \right)}.}}$

FIG. 6 shows the neurotoxicity of Aβ (1-40) in the absence or presenceof various peptides of the present invention. In this experiment theall-[D]-Lys-Lys-Leu-Val-Phe-Phe-Ala (SEQ. ID NO: 2) peptide is a morepotent inhibitor of Aβ neurotoxicity than theall-[L]-Lys-Lys-Leu-Val-Phe-Phe-Ala peptide in the cytoprotection assay.

FIG. 7 uses the propidium iodide assay on primary cortical neurons.Briefly, fetal rat primary cortical neurons are isolated and culturedaccording to Durkin, J. P. et al., J. Neurochem., 66:951-962, 1996.Neurons are plated in a 48 well microplate. 7 days after plating theneuronal culture media is supplemented with B27 (Life Technologies, Datasheet form No. 3755). A mixture of Aβ and test peptide is added to thecortical neurons for 3 days at 37° C. and 5% CO2.

Cell viability is then visually assessed as the proportion ofphase-bright cells that exclude propidium iodide, since only dead cellstake up propidium iodide.

FIG. 7 shows the potent cytoprotective activity ofall-[D]-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:3). This experiment shows thepotent cytoprotective activity of all-[D]-Lys-Leu-Val-Phe-Phe-Alacompared to Congo red, which is a known cytoprotective agent andcompared to the absence of any cytoprotective agent (Aβ alone).

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

1. A peptide for the prevention or treatment of an amyloidosis disorderin a human, said peptide comprising a sequence of [D]-amino acidsselected from the group consisting of: (i) Xaa₁-Xaa₂-Xaa₃-Xaa₄ and (ii)retro-isomer sequences of Xaa₁-Xaa₂-Xaa₃-Xaa₄, wherein: Xaa₁ is Lys;Xaa₂ is Ile, Lys-Leu, Phe, Ala, Val, or Leu; Xaa₃ is Val; and Xaa₄ isPhe-Phe-Ala, Phe-Phe-Ala-NH₂, Phe-Phe-Ala-Gln, or Phe-Phe-Ala-Gln-NH₂;with the proviso that Xaa₁-Xaa₂-Xaa₃-Xaa₄ is notLys-Leu-Val-Phe-Phe-Ala.
 2. The peptide of claim 1, wherein saidsequence of [D]-amino acids is selected from the group consisting of:Lys-Ile-Val-Phe-Phe-Ala; (SEQ ID NO:1) Lys-Lys-Leu-Val-Phe-Phe-Ala; (SEQID NO:2) Lys-Phe-Val-Phe-Phe-Ala; (SEQ ID NO:4) Lys-Ala-Val-Phe-Phe-Ala;(SEQ ID NO:7) Lys-Val-Val-Phe-Phe-Ala; (SEQ ID NO:9)Lys-Ile-Val-Phe-Phe-Ala-NH₂; (SEQ ID NO:10) Lys-Phe-Val-Phe-Phe-Ala-NH₂;(SEQ ID NO:12) Lys-Ala-Val-Phe-Phe-Ala-NH₂; (SEQ ID NO:15)Lys-Val-Val-Phe-Phe-Ala-NH₂; (SEQ ID NO:17) Lys-Leu-Val-Phe-Phe-Ala-Gln;(SEQ ID NO:18) Lys-Leu-Val-Phe-Phe-Ala-Gln-NH₂; (SEQ ID NO:19)

and retro-isomer sequences thereof.
 3. The peptide of claim 1, whereinsaid peptide is associated to a pharmaceutically acceptable carrier. 4.The peptide of claim 1, wherein said amyloidosis disorder is Alzheimer'sdisease.
 5. The peptide of claim 1, wherein said peptide is forpreventing the development of Alzheimer's disease in a human.
 6. Thepeptide of claim 1, wherein said peptide is for arresting thedevelopment of Alzheimer's disease in a human.
 7. A peptide comprising asequence of [D]-amino acids, wherein said sequence [D]-amino acids isLys-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:2) or the retro-isomer sequencethereof.
 8. A medicament for the prevention or treatment of anamyloidosis disorder in a human, said medicament comprising a peptide asdefined in claim
 1. 9. The medicament of claim 8, wherein saidmedicament is for preventing or for arresting the development ofAlzheimer's disease in a human.
 10. A method for blocking or preventingan amyloidosis disorder in a patient, said method comprisingadministering to said patient a peptide comprising a sequence of[D]-amino acids selected from the group consisting of: (i)Xaa₁-Xaa₂-Xaa₃-Xaa₄ and (ii) retro-isomer sequences ofXaa₁-Xaa₂-Xaa₃-Xaa₄, wherein: Xaa₁ is Lys; Xaa₂ is Ile, Lys-Leu, Phe,Ala, Val, or Leu; Xaa₃ is Val; and Xaa₄ is Phe-Phe-Ala, Phe-Phe-Ala-NH₂,Phe-Phe-Ala-Gln, or Phe-Phe-Ala-Gln-NH₂; with the proviso thatXaa₁-Xaa₂-Xaa₃-Xaa₄ is not Lys-Leu-Val-Phe-Phe-Ala.
 11. The method ofclaim 10, wherein said sequence of [D]-amino acids is selected from thegroup consisting of: Lys-Ile-Val-Phe-Phe-Ala; (SEQ ID NO:1)Lys-Lys-Leu-Val-Phe-Phe-Ala; (SEQ ID NO:2) Lys-Phe-Val-Phe-Phe-Ala; (SEQID NO:4) Lys-Ala-Val-Phe-Phe-Ala; (SEQ ID NO:7) Lys-Val-Val-Phe-Phe-Ala;(SEQ ID NO:9) Lys-Ile-Val-Phe-Phe-Ala-NH₂; (SEQ ID NO:10)Lys-Phe-Val-Phe-Phe-Ala-NH₂; (SEQ ID NO:12) Lys-Ala-Val-Phe-Phe-Ala-NH₂;(SEQ ID NO:15) Lys-Val-Val-Phe-Phe-Ala-NH₂; (SEQ ID NO:17)Lys-Leu-Val-Phe-Phe-Ala-Gln; (SEQ ID NO:18)Lys-Leu-Val-Phe-Phe-Ala-Gln-NH₂; (SEQ ID NO:19)

and retro-isomer sequences thereof.
 12. The method of claim 10, whereinsaid peptide is associated to a pharmaceutically acceptable carrier. 13.The method of claim 10, wherein said patient is in need of a treatmentagainst an amyloidosis disorder.
 14. The method of claim 10, whereinsaid amyloidosis disorder is Alzheimer's disease.
 15. The method ofclaim 10, wherein said method is for preventing the development ofAlzheimer's disease.
 16. The method of claim 10, wherein said method isfor arresting the development of Alzheimer's disease.
 17. A method forpreventing or for arresting the development of Alzheimer's disease in ahuman, said method comprising administering to a patient a peptidecomprising a sequence of [D]-amino acids, wherein said sequence isLys-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:2) or the retro-isomer thereof.